Stabalized Glycosaminoglycan Preparations and Related Methods

ABSTRACT

Compositions comprising a glycosaminoglycan (e.g., a hyaluronan, hyaluronic acid, hyaluronate, sodium hyaluronate, dermatan sulfate, karatan sulfate, chondroitin 6-sulfate, heparin, etc.) in combination with at least one component selected from; i) polyglycols (e.g., polyethylene glycol), ii) long chain hydroxy polyanionic polysaccharides (e.g., dextran, sodium alginate, alginic acid, propylene glycol alginate, carboxymethyl cellulose and carboxyethyl cellulose, hydroxyl ethyl starch, hydroxyl propyl methyl cellulose, hydroxy propyl ethyl cellulose, hydroxy propyl cellulose, methyl cellulose, polylysine, polyhistidine, polyhydroxy proline, poly ornithine, polyvinyl pyrolidone, polyvinyl alcohol, chitosan, etc.) and iii) long chain Nitrogen containing polymers (e.g., Polylysine, Polyvinylpyrrolidone, and polyvinyl alcohol). The invention also includes methods for using such compositions (e.g., as substance delivery materials, tissue fillers or bulking agents, as moistening or hydrating agents, etc.)

RELATED APPLICATION

This application is a continuation of copending U.S. patent applicationSer. No. 13/648,037 filed Oct. 9, 2012 and issuing as U.S. Pat. No.8,697,671 on Apr. 15, 2014, which is a division of U.S. patentapplication Ser. No. 11/940,171 filed Nov. 14, 2007 and issuing on Oct.16, 2012 as U.S. Pat. No. 8,288,362, which is a continuation in part ofU.S. patent application Ser. No. 11/126,075 filed May 9, 2005 and issuedas U.S. Pat. No. 7,544,671, which claims priority to U.S. ProvisionalPatent Application No. 60/569,407 filed on May 7, 2004, the entiredisclosures of each such earlier-filed application being expresslyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the fields of chemistry,pharmaceutical formulation and medicine and more particularly tostabilized glycosaminoglycan compositions and their methods ofmanufacture and use.

BACKGROUND OF THE INVENTION

Hyaluronan is part of a group of polysaccharides known asglycosaminoglycans. In particular, hyaluronan is a mucopolysaccharidethat occurs naturally in the bodies of humans and other animals. Theterm hyaluronan encompasses hyaluronic acid as well as salts ofhyaluronic acid, such as sodium hyaluronate. The term hyaluronate refersto the conjugate base of hyaluronic acid. Hyaluronan is the polyanionicform of hyaluronic acid, which exists in vivo.

In general, glycosaminoglycans are made up of repeating disaccharideunits containing a derivative of an aminosugar. The repeatingdisaccharide unit of hyaluronan consists of alternating glucuronic acidand N-acetylglucosamine units, which are repeated over and over to formlong chains. Each repeating disaccharide unit has one carboxylate group,four hydroxyl groups, and an acetamido group. Hyaluronan differs fromthe other major glycosaminoglycans in that it does not have sulfategroups. The chemical structure of hyaluronan is as follows:

Hyaluronan in the extracellular matrix of various body tissues. Innormal physiological states hyaluronan molecules form random coils inthe nature of helical ribbons that are stiffened by hydrogen bonds andsolvent interactions. The axial hydrogen atoms are relatively non-polarwhile the equatorial side chains are relatively polar, thereby creatingthe twisting ribbon structure.

Hyaluronan is synthesized in the body by many types of cells and tendsto collect in extracellular spaces where acts as a scaffold for aggrecanself-assembly, thereby combining with other constituents to formsupportive or protective networks around the cells. Hyaluronan ispresent in many body fluids and tissues and is found in relatively highconcentrations in vitreous humor and synovial fluid.

Hyaluronan is highly lubricious, hydrophilic and exhibits uniquerheological properties. The unique rheology of hyaluronan is believed tobe due at least in part to the fact that the hyaluronan polymer coilsbecome entangled with each other at low concentrations and exhibitshear-dependent viscosity at high concentrations. For example, a 1%solution of hyaluronan may exist as a gelatinous mass under ambientconditions but, when compressed, will become less viscous and easilyflowable such that it may be injected through a hypodermic needle.Because of this unique rheological behavior, hyaluronan has beenreferred to as a “pseudo-plastic” material. The hydrophilic nature ofhyaluronan is believed to be a function of the fact that hyaluronanforms stiffened helical ribbons as described above. Each such helicalribbon is configured such that it may trap substantial amounts of water(e.g., approximately 1000 times its weight in water).

Hyaluronan has a wide variety of medical and non-medical applications.For example, hyaluronan solutions make excellent lubricants and mayallow tissue surfaces to slide over one another. Thus, hyaluronanpreparations are sometimes applied to tissues to promote healing and/orto reduce the potential for postoperative adhesion formation. One of itsimportant biological roles is to provide beneficial effects on woundhealing in the skin and eyes.

Recently, hyaluronan has been found to enhance corneal epithelialhealing and corneal reepithelialization for non-infectious cornealerosion. These beneficial effects can be extended to the management ofdry eye syndrome, allergic conjunctivitis, and contact lens wear.

For example, dry eye is a syndrome in which inadequate tear productionand inappropriate tear composition causes the cornea and conjunctivaimproper wetting. Untreated dry eye can be further deteriorated toproduce more severe epithelial erosion, strands of epithelial cells, dryspots on the cornea. These can be complicated further by microbialinfection. Thus, an early medical management for the dry eye syndromewould be highly desirable. Such an early treatment of the dryness andirritation of the eye by the use of hyaluronan could be very effectiveand beneficial medical management of the dry eye.

Additionally, it has been known for a long time that contact lenseswhich have adsorption of cellular debris, mucus materials, lipids andproteins from the eye can cause irritation and/or infection of the eye.Thus, a biocompatible lubricant, particularly hyaluronan can providebeneficial effects to prevent the deposit from forming in its earlystage of deposit formation on the contact lenses in the eye.

As indicated above, beneficial effects of hyaluronan for the health ofthe eye are great; however, use of hyaluronan has been rather limiteddue to its chemical instability losing its viscosity and lubricity inaqueous solution.

The prior art has included a number of stabilized hyaluronic acid gelsuseable for cosmetic tissue bulking. For example, RESTALYNE® InjectableGel; (Medicis Pharmaceutical Corporation, Scottsdale, Ariz.) is asterile gel of stabilized sodium hyaluronate. Also, PERLANE® injectablegel (Medicis Pharmaceutical Corporation, Scottsdale, Ariz.) is a sterilegel of hyaluronic acid chemically cross-linked with BODE, stabilized andsuspended in phosphate buffered saline at pH=7 and concentration of 20mg/mL.

Additionally, the prior art has included a number of hyaluronic acidcontaining viscoelastic preparations used in cataract surgery. Forexample, VISCOAT® viscoelastic solution (Alcon) contains purified mediummolecular weight sodium chondroitin sulfate and sodium hyaluronateformulated to a viscosity of 40,000±20,000 cps at shear rate of 2 sec⁻¹,25° C. VISCOAT® viscoelastic solution is relatively unstable at roomtemperature and its manufacturer recommends that it be stored at 2°-8°C. (36°-46° F.) and warmed to room temperature just prior to use. Also,the ProVisc® viscoelastic solution is a cohesive viscoelastic containinga high molecular weight, non-inflammatory highly purified fraction ofsodium hyaluronate, dissolved in physiological sodium chloride phosphatebuffer. It is also relatively unstable at room temperature and itsmanufacturer recommends that it be stored at 2′-8° C. (36°-46° F.) andwarmed to room temperature just prior to use. The VISCOAT® viscoelasticsolution and the ProVisc® viscoelastic solution are sometimes used andformulated in combination. For example, the DisCoVisc® OphthalmicViscosurgical Device (Alcon) is product which combines the VISCOAT®viscoelastic solution and the ProVisc® viscoelastic solution in a singlepre-filled syringe and the DuoVisc® Viscoelastic system (Alcon) is aproduct that provides VISCOAT® viscoelastic solution and the ProVisc®viscoelastic solution in separate pre-filled syringes. Like theirindividual components, the DisCoVisc® and DuoVisa® products must also bestored at 2°-8° C. (36°-46° F.) and warmed to room temperature justprior to use.

There remains a need in the art for the development of new stabilizedglycosaminoglycan materials, such as hyaluronic acid or salts ofhyaluronic acid, that are stable when stored at room temperature andwhich exhibit desirable rheological properties.

SUMMARY OF THE INVENTION

The present invention provides compositions that compriseglycosaminoglycans, such as hyaluronic acid or another hyaluronan, incombination with a stabilizing and/or viscosity increasing agent. Thepresent invention also provides methods for using such compositions.

In accordance with the invention, there are provided compositions thatcomprise a glycosaminoglycan (e.g., hyaluronic acid or a hyaluronic acidsalt) in combination with at least one agent selected from the groupconsisting of i) polyglycols (e.g., polyethylene glycol), ii) HydroxyPolyanionic Polysaccharides (e.g., dextran, sodium alginate, alginicacid, propylene glycol alginate, carboxymethyl cellulose andcarboxyethyl cellulose, hydroxyl ethyl starch, hydroxyl propyl methylcellulose, hydroxy propyl ethyl cellulose, hydroxy propyl cellulose,methyl cellulose, polylysine, polyhistidine, polyhydroxy praline, polyornithine, polyvinyl pyrolidone, polyvinyl alcohol, chitosan, etc.) andiii) long chain Nitrogen containing polymers (e.g., Polylysine,Polyvinylpyrrolidone, and polyvinyl alcohol). Such compositions may beprepared in aqueous solution having a pH of from about 5.0 to about 9.0,preferably pH 7.0-7.4.

In accordance with one embodiment of the invention, a hyaluronan such ashyaluronic acid may be combined with at least one polyglycol in a ratio,and under conditions, which result in the preparation retaining theviscosity and lubricity of the hyaluronan substantially longer than ifit had not been combined with the polyglycol. Any suitable hyaluronanlike, hyaluronic acid sodium salt and any suitable polyglycol like,polyethylene glycol, including long chain hydroxy polyanionicpolysaccharides (eg., sodium alginate, alginic acid, propylene glycolalginate, carboxymethyl cellulose, carboxyethyl cellulose, etc.), butnot polyanionic polysaccharides (eg., Dextran sulfates), long chainnitrogen containing polymers (eg., polylysine, polyhistidine,polyhydroxy proline, polyornithine, polyvinylpyrrolidone, etc.), hydroxypolysaccharides (eg., hydroxyethyl starch, dextran, hydroxypropylmethylcellulose, Hydroxy Propyl Cellulose, Methyl Cellulose etc.), nitrogencontaining hydroxy polysaccharides (eg., Chitosan, etc.), andpolyhydroxy linear polymers (eg., polyvinyl alcohol, polyhydroxyproline, etc.) may be used, particularly, the long chain hydroxypolyanionic polysaccharides are not only stabilizing the hyaluronan butalso increasing viscosity and lubricity of the hyaluronansynergistically. For example, see Example 1. In some cases, thepreparation may include other active or inactive ingredients orreactants including, but not limited to, drugs, cosmetics,preservatives, pH adjusting agents, tonicity adjusting agents,thickening or gelling agents, water, coloring agents, fragrance, etc.The stabilized hyaluronan preparations of this invention may be liquidsolutions, gels, creams, or any other useable forms. The stabilizedhyaluronan preparations of this invention may be used for a variety ofmedical and non-medical (e.g., household or industrial) applications,including topical administration to the eye (e.g., to moisturize theeye, treat dry eye, promote corneal healing, facilitatereepithelialization for non-infectious corneal erosion, management ofdry eye syndrome, allergic conjunctivitis, and contact lens wear, etc.),topical administration (e.g., to moisturize the skin, to treat dry skinor dermatological disorders), lubrication or body tissues or bodyorifices, lubrication of devices (e.g., catheters, scopes, instruments,etc.), application to tissues during surgery to deter post-surgicaladhesion formation, subcutaneous injection for removing wrinkles inaddition as vehicles and active drug delivery depot.

Further in accordance with the invention, there are provided methods formanufacturing hyaluronan preparations wherein hyaluronan is combinedwith a polyglycol. The hyaluronan may be combined with the polyglycol ina ratio and under conditions that result in reaction without chemicalstructural changes (e.g., chemical conformational changes via complexformation) between the hyaluronan and the polyglycol such that remainsstable for an extended period of time (e.g., 2 years or more) at roomtemperature.

Further aspects, details and embodiments of the present invention willbe understood by those of skill in the art upon reading the detaileddescription and examples set forth herebelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of viscosity vs. shear rate for room temperature, pH5.2 solutions of: 0.5% carboxy methyl cellulose (CMC), 0.15% hyaluronicacid having a molecular weight of 2 million Daltons (2 MDa HA) and 0.15%2 MDa HA+0.5% CMC.

FIG. 1B is a graph of viscosity vs. shear rate for room temperature, pH5.2 solutions of: 0.5% carboxy methyl cellulose (CMC) alone, 0.15%hyaluronic acid having a molecular weight of 1.5 million Daltons (1.5MDa HA) alone and 0.15% 1.5 MDa HA+0.5% CMC.

FIG. 1C is a graph of viscosity vs. shear rate for room temperature. pH5.2 solutions of: 0.5% carboxy methyl cellulose (CMC) alone, 0.15%hyaluronic acid having a molecular weight of 1 million Daltons (1 MDaHA) alone and 0.15% 1 MDa HA+0.5% CMC.

FIG. 2 is a graph of viscosity vs. shear rate for room temperaturesolutions of: 0.15% 2 MDa HA+0.5% CMC @ pH 7.2, 0.15% 2 MDa HA+0.5% CMC@ pH 5.2, 0.15% 2 MDa HA @ pH 7.2 and 0.15% 2 MDa HA @ pH 5.2.

FIG. 3 is a graph of viscosity vs. shear rate for 25° C., pH 7.2solutions of: 0.15% 2 MDa HA+0.5% hydroxy propyl methyl cellulose(HPMC), 0.5% HPMC and 0.15% 2 MDa HA.

FIG. 4 is a graph of viscosity vs. shear rate for 25° C., pH 7.2solutions of: 0.15% 2 MDa HA+0.5% sodium alginate (NaALG), 0.5% NaALGand 0.15% 2 MDa HA.

FIG. 5 is a graph of viscosity vs. shear rate for 25° C., pH 7.2solutions of: 0.15% 2 MDa HA+0.5% propylene glycol alginate (PG-ALG),0.5% PG-ALG and 0.15% 2 MDa HA.

FIG. 6 is a graph of viscosity vs. shear rate for 25° C., pH 7.2solutions of: 0.15% 2 MDa HA+0.5% CMC, 0.5% CMC and 0.15% 2 MDa HA.

DETAILED DESCRIPTION

The following detailed description is intended to describe some, but notnecessarily all, examples or embodiments of the invention. No effort hasbeen made to exhaustively describe all possible examples and embodimentsof the invention. Thus, the contents of this detailed description shallnot limit the scope of the invention in any way.

Polyglycol Stabilized Hyaluronans

In accordance with the present invention, there are providedcompositions comprising a hyaluronan combined with a polyglycol, whereinthe properties of the hyaluronan (e.g., viscosity and lubricity) aremaintained for a prolonged period of time. Thus, polyglycols may beadded to or included in various hyaluronan preparations to prolong theshelf stability and usefulness of such preparations.

A polyglycol is defined as a polyhydric alcohol of a monomeric glycol.Polyethylene Glycols (PEGs) are a family of linear, water-solublepolyglycols. PEGs are formed by polymerization of ethylene oxide. Thegeneralized formula for polyethylene glycol is:

H—(OCH₂CH₂)_(n)—OH

where “n” is the average number of repeating oxyethylene groups.

Using the methods of the present invention, hyaluronan can be complexedwith a PEG to form hyaluronan preparations that remain stable at roomtemperature for extended periods of time (e.g., 2 years or more) withoutsubstantial chemical break down of the hyaluronan and resultant changein viscosity and lubricity.

In preparations of the present invention wherein hyaluronan is combinedwith a polyglycol (e.g., PEG), the polyglycol may preferably have anaverage molecular weight in the range of about 200 to about 35,000 andmore preferably, in at least some applications, an average molecularweight in the range of about 6000 to about 8000.

Also, in preparations of the present invention wherein hyaluronan iscombined with a polyglycol (e.g., PEG), the hyaluronan may preferablyhave an average molecular weight in the range of about 2×10³ to about5×10⁶ and more preferably, in at least some applications, an averagemolecular weight in the range of about 2×10⁵-3×10⁶.

Also, in preparations of the present invention wherein hyaluronan iscombined with a polyglycol (e.g., PEG), the weight ratio of hyaluronanto polyglycol may be in the range of from about 0.1:1 to about 10:1 andmore preferably in at least some applications such weight ratio ofhyaluronan to polyglycol may be in the range of from about 1:2 to about1:10.

Also, in preparations of the present invention wherein hyaluronan iscombined with a polyglycol (e.g., PEG) and any other optional componentsexamples of which are set forth in the formulations shown in Examples 1and 2 below, the concentration of hyaluronan in the preparation may bein the range of about 0.01% by weight to about 10% by weight.

Also, in preparations of the present invention wherein hyaluronan iscombined with a polyglycol (e.g., PEG) and any other optional componentsexamples of which are set forth in the formulations shown in Examples 1and 2 below, the pH of the preparation may be in the range of from about5.0 to about 9.5 or more preferably in at least some applications, fromabout 7.0 to about 7.4. Appropriate acidifying and/or alkaline (e.g.,buffering) agents may be added in accordance with procedures well knownin the art to adjust the pH of the preparation as needed or desired.

Also, in preparations of the present invention wherein hyaluronan iscombined with a polyglycol (e.g., PEG) and any other optional componentsexamples of which are set forth in the formulations shown in Examples 1and 2 below, the tonicity of the preparation may preferably be in therange of about 200 mOsm to about 340 mOsm. Hyperosmolar and/orhypoosmolar agents (e.g., manitol, water, etc.) may be added inaccordance with procedures well known in the art to adjust the tonicityof the preparation as needed or desired.

EXAMPLE 1 A Stabilized Hyaluronan Preparation

In this example, a liquid hyaluronan preparation is prepared bycombining and mixing the components of the following formulation at roomtemperature:

Hyaluronic Acid Sodium Salt 0.15% Polyethyleneglycol (PEG 8000) 0.50%Boric Acid 0.20% Sodium Chloride 0.58% Postassium Chloride 0.14% CalciumChloride Dihydrate 0.02% Magnesium Chloride Hexahydrate 0.011%  SodiumChorite/Hydrogen Peroxide 0.06% Purified Water Q.S to 100 mL.

This results in a viscous liquid preparation that is suited for a widevariety of medical or non-medical uses, including use as a lubricant ormoisturizing agent, for topical administration to the skin, mucousmembranes or eyes, or as a carrier for cosmetics, pharmaceuticals orother agents.

EXAMPLE 2 Stability Comparison

In this example, hyaluronan compositions were prepared under ambient,room temperature conditions according to Formulations I and II, asfollows:

Formula I: Formula II: Hyaluronic Acid Sodium 0.15% Hyaluronic AcidSodium 0.15% Salt Salt — Polyethyleneglycol  0.5% (PEG 8000) Boric Acid 0.2% Boric Acid  0.2% Sodium Chloride 0.58% Sodium Chloride 0.58%Postassium Chloride 0.14% Postassium Chloride 0.14% Calcium Chloride0.02% Calcium Chloride 0.02% Dihydrate Dihydrate Magnesium Chloride0.11% Magnesium Chloride 0.11% Hexahydrate Hexahydrate Sodium Chorite/0.06% Sodium Chorite/ 0.06% Hydrogen Peroxide Hydrogen Peroxide PurifiedWater Q.S to 100 Purified Water Q.S to 100 mL. mL.

Both Formulation 1 and Formulation 2 provide a lubricious liquidsolution as described in Example 1 above. However, when stored at roomtemperature, the preparation of Formula I looses substantial viscosityand becomes substantially less lubricious within few weeks. In contrast,the preparation of Formula II remains stable and does not undergo anysubstantial change in viscosity or lubricity for at least two (2) years.

Long Chain Hydroxy Polyanionic Polysaccharide Stabilized Hyaluronans andOther Glycosaminoglycans

Glycosaminoglycans (Including Hyaluronic acid) are important substancesin connective tissues in determining the viscoelastic properties ofjoints and of other structures that are subject to mechanicaldeformation. Hyaluronic acid is one of the major glycosaminoglycans thatoccur in many living substances as synovial fluid. Hyaluronic acid is amajor component of the extracellular matrix and contributes to cellularmigration and proliferation and neuronal morphogenesis. Hyaluronic acidalso acts as a lubricant and shock absorber in joints, in the vitreousbody of the eye and other parts of the body.

One drawback of administering exogenous hyaluronic acid for therapeuticor other biomedical purposes is that hyaluronic acid degrades veryrapidly and consequently loses its viscosity and its lubricity. Thislimits the many biomedical applications of the hyaluronic acid. Some ofthe noteworthy therapeutic and other biomedical applications ofhyaluronic acid have included its use in tissue grafting, catheterlubrication for insertion into human bodies, scope and endoscopelubrication, skin grafting, dermal wound healing, cell culture,bioengineering and cell bioengineering, eye and joint lubrication,vitreous humor replacement, artificial tears for dry eyes, contact lensstorage and wear, corneal wound healing, treatment of allergicconjunctivitis, as a viscoelastic agent in cataract surgery, as anadditive in cosmetics and personal care products, as a drug deliveryvehicle, etc.

Applicants have discovered that hyaluronic acid can also be stabilizedwith Long chain hydroxy polyanionic polysaccharides (e.g., sodiumalginate, alginic acid, propylene glycol alginate, carboxymethylcellulose and carboxyethyl cellulose, etc.) in an aqueous solution of pH5.0-9.0. It has been further discovered that the viscosity of thehyaluronic acid (HA) in aqueous solution at pH 5.0-9.0 can be increasedby an order of magnitude or more with the addition of one or more longchain hydroxy polyanionic polysaccharides and such hyaluronic Acid+longchain hydroxy polyanionic polysaccharides possess a non-Newtonian fluidcharacteristics (shear thinning effects) (see FIG. 1, 2, 3, 4).

EXAMPLE 3 Comparison of Viscosity Enhancement and Shear Thinning Effects

In this example, the viscosities of various test samples were measuredat shear rates ranging from 1-200 1/sec. The compositions of the testsamples were as follows:

Sample Sample Composition Sample A 0.15 g of 1 MDa Sodium (1 MDa HAAlone/pH 5.2.) Hyaluronate in 100 mL of the borate buffer solution @ pH5.2 Sample B 0.15 g of 1.5 MDa Sodium (1.5 MDa HA Alone/pH 5.2)Hyaluronate in 100 mL of the borate buffer solution @ pH 5.2 Sample C0.15 g of 2 MDa Sodium (2 MDa HA Alone/pH 5.2) Hyaluronate in 100 mL ofthe borate buffer solution @ pH 5.2 Sample D 0.15 g of 2 MDa Sodium (2MDa HA Alone/pH 7.2) Hyaluronate in 100 mL of the borate buffer solution@ pH 7.2 Sample E 0.5 g of CMC in 100 mL of the (CMC Alone/pH 5.2)borate buffer solution @ pH 5.2 Sample F 0.5 g of CMC in 100 mL of the(CMC Alone/pH 7.2) borate buffer solution @ pH 7.2 Sample G 0.15 g of 1MDa Sodium (1 MDa HA + CMC/pH 5.2) Hyaluronate + 0.5 g of CMC in 100 mLof the borate buffer solution 0.15 g of 2 MDa Sodium Hyaluronate in 100mL of the borate buffer solution @ pH 5.2 Sample H 0.15 g of 15M Sodium(1.5 MDa HA + CMC/pH 5.2) Hyaluronate + 0.5 g of CMC in 100 mL of theborate buffer solution 0.15 g of 2 MDa Sodium Hyaluronate in 100 mL ofthe borate buffer solution @ pH 5.2 Sample I 0.15 g of 2 MDa Sodium (2MDa HA + CMC/pH 5.2) Hyaluronate + 0.5 g of CMC in 100 mL of the boratebuffer solution @ pH 5.2 Sample J 0.15 g of 2 MDa Sodium (2 MDa HA +CMC/pH 7.2) Hyaluronate + 0.5 g of CMC in 100 mL of the borate buffersolution @ pH 7.2 Sample K 0.5 g HPMC in 100 mL of the (HPMC Alone/pH7.2) borate buffer solution @ pH 7.2 Sample L 0.15 g of 2 MDa Sodium (2MDa HA + HPMC/pH 7.2) Hyaluronate + 0.5 g of HPMC in 100 mL of theborate buffer solution @ pH 7.2 Sample M 0.5 g NaALG in 100 mL of the(NaALG Alone/pH 7.2) borate buffer solution @ pH 7.2 Sample N 0.15 g of2 MDa Sodium (2 MDa HA + NaALG/pH 7.2) Hyaluronate + 0.5 g of HPMC in100 mL of the borate buffer solution @ pH 7.2 Sample O 0.15 g of 2 MDaSodium (2 MDa HA + Pluronic Hyaluronate + 0.5 g of F127/pH 5.2) PluronicF127 in 100 mL of the borate buffer solution @ pH 5.2 Sample P 0.15 g of2 MDa Sodium (2 MDa HA + PEG/pH 5.2) Hyaluronate + 0.5 g of PluronicF127 in 100 mL of the borate buffer solution @ pH 5.2

Each sample was prepared in a borate buffer solution. The formulation ofthis borate buffer solution was as follows:

Component Amount Boric Acid 0.20 g Sodium Chloride 0.58 g PotassiumChloride 0.14 g Calcium Chloride Dihydrate 0.02 g Magnesium ChlorideHexahydrate 0.011 g  Purified Water Q.S. to 100 mL

To prepare the buffer, 1 g of boric acid, 2.9 g of Sodium chloride, 0.7g of potassium chloride, 0.1 g of calcium chloride dihydrate and 0.055 gmagnesium chloride hexahydrate were dissolved in distilled water. Thesolution was diluted to 500 mL. The pH of this buffer solution wasadjusted to either 5.2 or 7.2 at room temperature, as indicated.

To insure samples were thoroughly dissolved and well mixed, stocksolutions of the polymers in borate buffer were prepared when possible.For hyaluronic acids, 2% stock solutions were used. All stock solutionswere vortex mixed and then placed on a shaker until clear andhomogeneous.

Hyaluronic acid samples (0.15% HA) were prepared by dilutingapproximately 0.75 g of the HA stock solution to 10 g using the boratebuffer. Again, samples were vortex mixed and placed in a shaker untilthe solutions were clear and homogeneous.

Viscosity measurements were performed on an AR 1000 rheometer (T AInstruments) using a cone and plate geometry. The cone was 6 cm indiameter with an angle of 1 degree and a truncation gap of 29 mm. Therange of torques applied was adjusted to cover shear rates ranging (atminimum) from 25 sec⁻¹ to 160 sec⁻¹. Shear rates were first increasedand then during the experiment. A new sample was loaded for eachexperiment.

FIG. 1A is a graph showing viscosity vs. shear rate for samples C, E andI. As shown, at pH 5.2, the viscosity of Sample I (2 MDa HA+CMC) wassynergistically increased over the viscosity of Samples C (2 MDa HAalone) and E (CMC alone).

FIG. 1B is a graph showing viscosity vs. shear rate for samples B, E andH. As shown, at pH 5.2, the viscosity of Sample H (1.5 MDa HA+CMC) wassynergistically increased over the viscosity of Samples B (1.5 MDa HAalone) and E (CMC alone),

FIG. 1C is a graph showing viscosity vs. shear rate for samples A, E andG. As shown, at pH 5.2, the viscosity of Sample G (1 MDa HA+CMC) wassynergistically increased over the viscosity of Samples A (1 MDa HAalone) and E (CMC alone).

Also, when FIGS. 1A-1C are viewed in comparison, it is apparent that theviscosity enhancing effect of CMC was most pronounced in the samplecontaining 2 MDa HA (Sample K) and least pronounced in the samplecontaining 1 MDa HA (Sample G). Thus, the viscosity enhancing effect ofCMC is greater with higher molecular weight HA.

FIG. 2 is a graph showing viscosity vs. shear rate for samples C. D, Iand J. The viscosities of samples D (2 MDa HA alone at neutral pH 7.2)and J (2 MDa HA+CMC at neutral pH 7.2) were greater than the viscositiesof samples C (2 MDa HA alone at alkaline pH 5.2) and 1 (2 MDa HA+CMC atalkaline pH 5.2). Thus, the viscosity of HA, whether alone or incombination with CMC, increases as pH changes from alkaline (5.2) toneutral (7.2).

FIG. 3 is a graph showing viscosity vs. shear rate for samples D, K andL. These data indicate that, at pH 7.4, the viscosity of Sample L (2 MDaHA+HPMC) was synergistically increased over the viscosities of Samples D(2 MDa HA alone) and K (HPMC alone). Thus, the addition of 0.5% HPMCcaused a supraadditive (e.g., synergistic) increase in viscosity of theHA.

FIG. 4 is a graph showing viscosity vs. shear rate for samples D, M andN. These data indicate that, at pH 7.4, the viscosity of Sample N (2 MDaHA+NaALG) was synergistically increased over the viscosities of SamplesD (2 MDa HA alone) and M (Na-ALG alone). Thus, the addition of 0.5%Na-ALG caused a supraadditive (e.g., synergistic) increase in viscosityof the HA.

FIG. 5 is a graph showing viscosity vs. shear rate for samples D, N andO. These data indicate that, at pH 7.4, the viscosity of Sample O (2 MDaHA+PG-ALG) was synergistically increased over the viscosities of SamplesD (2 MDa HA alone) and N (PG-ALG alone). Thus, the addition of 0.5%PG-ALG caused a supraadditive (e.g., synergistic) increase in viscosityof the HA.

FIG. 6 is a graph showing viscosity vs. shear rate for samples D, F andJ. These data indicate that, at pH 7.4, the viscosity of Sample J (2 MDaHA+CMC) was synergistically increased over the viscosities of Samples D(2 MDa HA alone) and F (CMC alone). Thus, the addition of 0.5% CMCcaused a supraadditive (e.g., synergistic) increase in viscosity of theHA.

In the following table, the shear thinning grade ratio (STG ratio) wascalculated as the ratio of the viscosity at 160 sec to the viscosity at40 sec⁻¹ at 30° C., pH 5.2. The following table

Sample STG Ratio Sample C 1.42 (2 MDa HA Alone/pH 5.2) Sample I 1.50 (2MDa HA + CMC/pH 5.2) Sample P 1.53 (2 MDa HA + PEG/pH 5.2) Sample O 1.45(2 MDa HA + Pluronic F127/pH 5.2)

Pluronic F-127 is a registered trademark of BASF Corporation, FlorhamPark, N.J. It is a difunctional block copolymer surfactant terminatingin primary hydroxyl groups also known as Poloxamer 407.

As seen in the table above, at pH 5.2, the STG ratio is greater when 2MDa HA is combined CMC or PEG than for 2 MDa HA alone or in combinationwith a stabilizing amount of Pluronic F127. For example, an aqueous gelcontaining 30% CMC (a stabilizer) contained in a syringe fitted with a29 gauge needle cannot be injected through the 29 gauge needle at roomtemperature (or may be forced through that needle only with greatdifficulty). However, the addition of 0.15 g HA (0.15%) into the CMC gelmakes the gel become much thinner when a mechanical force is applied onit due to the shear thinning effects. Accordingly, the HA+CMC gel may beinjected through the 29 gauge needle easily using a standard syringe andstandard injection technique. This shear thinning property of the gel isof great value in preparations that are intended to be injected throughneedles or otherwise passed through small diameter lumens, passagewaysor openings. Examples of specific applications where these shearthinning effects will be of value will be in glycosaminoglycan gels(e.g., HA gels) and other preparations intended for use as substancedelivery depots/carriers or dermal/tissue fillers.

Accordingly, the hyaluronic acid/long chain hydroxy polyanionicpolysaccharides system becomes thinner as a mechanical force is appliedon it. Thus, it can be ideally used as an injectable formulation alone(e.g., as a viscoelastic agent or tissue filler/bulking agent) or may becombined with diagnostic or therapeutic agents (e.g., as a drug delivercarrier/depot) such that the diagnostic or therapeutic agents are thenreleased kinetically.

Glycosaminoglycans, like hyaluronic acid, are made up of disacchariderepeating units containing a derivative of an amino sugar, eitherglucosamine or galactosamine in which one of the sugars has a negativelycharged carboxylate or sulfate. Thus, glycosaminoglycans can bestabilized by the long chain hydroxyl polyanionic polysaccharides. Somemajor glycosaminoglycans are Dermatan Sulfate, Karatan Sulfate,Chondroitin 6-Sulfate and Heparin.

Glucosaminoglycans, like hyaluronic acid, are made up of disacchariderepeating units containing a derivative of an amino sugar, eitherglucosamine or galactosamine in which one of the sugars has a negativelycharged carboxylate or sulfate group. Thus, Aginic acid, Polyethyleneglycol alginate, CMC, etc. can also stabilize the glycosaminoglycans.

The term glycosaminoglycan as used herein includes but is not limited tohyaluronans, dermatan sulfate, keratan sulfate, chondroitin 6-sulphateand heparin.

In addition the stabilization of the above mentioned glycosaminoglycanscan be performed using Carboxyethyl Cellulose as well as many otherCarboxymethyl and Carboxyethyl cellulose compounds.

In summary, the combination of hyaluronic acid with a long chainhydroxyl polyanionic polysaccharides (e.g., Alginic acid, sodiumalginate, Propylene glycol alginate, dextran, CMC, etc.) results in acomposition having unique properties, including but not limited to:

-   -   1. Addition of the Long Chain Hydroxy Polyanionic        Polysaccharides stabilizes the unstable Hyaluronic acid        (Glycosaminoglycans)    -   2. Addition of the Long Chain Hydroxy Polyanionic        Polysaccharides increases the viscosity of Hyaluronic Acid        synergically in aqueous solution at pH 5.0-9.0 by an order of        magnitude-, or more.    -   3. Addition of the Long Chain Hydroxy Polyanionic        Polysaccharides enhances the shear thinning effects where the        enhancement is pronouncedly higher with a higher molar mass of        Hyaluronic Acid.    -   4. Addition of the hydroxy polysaccharides as well as nitrogen        containing hydroxyl polysaccharides and long chain nitrogen        containing polymers.    -   5. Polyhydroxy linear polymers.

The following are non-limiting examples of formulations of the presentinvention.

FORMULATION EXAMPLES

Formulation 1 Sodium Hyaluronate 0.001%-50.0% Sodium Alginate0.001%-50.0% Boric Acid 0.01%-1.0% Sodium Chloride  0.1%-10.0% PotassiumChloride  0.01%-10.0% Calcium Chloride dehydrate 0.001%-10.0% MagnesiumChloride hexahydrate 0.001%-1.0%  HCl or NaOH Adjust pH to 7.2 Purifiedwater, USP Q.S. to 100 mL

Formulation 2 Sodium Hyaluronate 0.001%-50.0% Alginic Acid 0.001%-50.0%Boric Acid 0.01%-1.0% Sodium Chloride  0.1%-10.0% Potassium Chloride 0.01%-10.0% Calcium Chloride dehydrate 0.001%-10.0% Magnesium Chloridehexahydrate 0.001%-1.0%  HCl or NaOH Adjust pH to 7.2 Purified water,USP Q.S. to 100 mL

Formulation 3 Sodium Hyaluronate 0.001%-50.0% Carboxymethylcellulose0.005%-40.0% Boric Acid 0.01%-1.0% Sodium Chloride  0.1%-10.0% PotassiumChloride  0.01%-10.0% Calcium Chloride dehydrate 0.001%-10.0% MagnesiumChloride hexahydrate 0.001%-1.0%  HCl or NaOH Adjust pH to 7.2 Purifiedwater, USP Q.S. to 100 mL

Formulation 4 Sodium Hyaluronate 0.001%-50.0% Propylene Glycol Alginate0.001%-50.0% Boric Acid 0.01%-1.0% Sodium Chloride  0.1%-10.0% PotassiumChloride  0.01%-10.0% Calcium Chloride dehydrate 0.001%-10.0% MagnesiumChloride hexahydrate 0.001%-1.0%  HCl or NaOH Adjust pH to 7.2 Purifiedwater, USP Q.S. to 100 mL

Formulation 5 Sodium Hyaluronate 0.001%-50.0% Hydroxy propyl methylcellulose 0.001%-50.0% Boric Acid 0.01%-1.0% Sodium Chloride  0.1%-10.0%Potassium Chloride  0.01%-10.0% Calcium Chloride dehydrate 0.001%-10.0%Magnesium Chloride hexahydrate 0.001%-1.0%  HCl or NaOH Adjust pH to 7.2Purified water, USP Q.S. to 100 mL

Formulation 6 Sodium Hyaluronate 0.001%-50.0% Sodium Alginate0.005%-40.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 7 Sodium Hyaluronate 0.001%-50.0% Alginic Acid 0.005%-40.0%Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% Sodium PhosphateDibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HCl or NaOHAdjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 8 Sodium Hyaluronate 0.001%-50.0% Propylene Glycol alginate0.005%-40.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 9 Sodium Hyaluronate 0.001%-50.0% Carboxymethylcellulose0.005%-40.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 10 Sodium Hyaluronate 0.001%-50.0% Hydroxy propyl methylcellulose 0.005%-40.0% Sodium Phosphate Monobasic, monohydrate0.01%-0.1% Sodium Phosphate Dibasic, anhydrous  0.02-1.0% SodiumChloride 0.01%-1.0% HCl or NaOH Adjust pH to 7.2 Purified water, USPQ.S. to 100 mL

Formulation 11 Sodium Hyaluronate 0.15%-3.0% Sodium Alginate 0.50%-15.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 12 Sodium Hyaluronate 0.15%-3.0% Alginic Acid  0.50%-15.0%Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% Sodium PhosphateDibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HCl or NaOHAdjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 13 Sodium Hyaluronate 0.15%-3.0% Propylene Glycol Alginate 0.50%-15.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 14 Sodium Hyaluronate 0.15%-3.0% Carboxymethylcellulose 0.50%-15.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 15 Sodium Hyaluronate 0.15%-3.0% Hydroxy propyl methylcellulose  0.50%-15.0% Sodium Phosphate Monobasic, monohydrate0.01%-0.1% Sodium Phosphate Dibasic, anhydrous  0.02-1.0% SodiumChloride 0.01%-1.0% HCl or NaOH Adjust pH to 7.2 Purified water, USPQ.S. to 100 mL

Formulation 16 Sodium Chondroitin Sulfate 0.001%-50.0% Alginic Acid0.001%-40.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 17 Sodium Chondroitin Sulfate 0.001%-50.0% Sodium alginate0.001%-40.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% SodiumPhosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HClor NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 18 Sodium Chondroitin Sulfate 0.001%-50.0% Propylene Glycolalginate 0.001%-40.0% Sodium Phosphate Monobasic, monohydrate 0.01%-0.1%Sodium Phosphate Dibasic, anhydrous  0.02-1.0% Sodium Chloride0.01%-1.0% HCl or NaOH Adjust pH to 7.2 Purified water, USP Q.S. to 100mL

Formulation 19 Sodium Chondroitin Sulfate 0.001%-50.0% Hydroxy propylmethyl cellulose 0.001%-40.0% Sodium Phosphate Monobasic, monohydrate0.01%-0.1% Sodium Phosphate Dibasic, anhydrous  0.02-1.0% SodiumChloride 0.01%-1.0% HCl or NaOH Adjust pH to 7.2 Purified water, USPQ.S. to 100 mL

Formulation 20 Sodium Hyaluronate 0.001%-50.0% Dextran 0.005%-40.0%Sodium Phosphate Monobasic, monohydrate 0.01%-0.1% Sodium PhosphateDibasic, anhydrous  0.02-1.0% Sodium Chloride 0.01%-1.0% HCl or NaOHAdjust pH to 7.2 Purified water, USP Q.S. to 100 mL

Formulation 21 Sodium Hyaluronate 0.001%-50.0% Dextran 0.005%-50.0%Boric Acid 0.01%-1.0% Sodium Chloride  0.1%-10.0% Potassium Chloride 0.01%-10.0% Calcium Chloride dehydrate 0.001%-10.0% Magnesium Chloridehexahydrate 0.001%-1.0%  HCl or NaOH Adjust pH to 7.2 Purified water,USP Q.S. to 100 mL

The invention has been described hereabove with reference to certainexamples or embodiments of the invention but that various additions,deletions, alterations and modifications may be made to those examplesand embodiments without departing from the intended spirit and scope ofthe invention. For example, any element or attribute of one embodimentor example may be incorporated into or used with another embodiment orexample, unless to do so would render the embodiment or exampleunsuitable for its intended use. All reasonable additions, deletions,modifications and alterations are to be considered equivalents of thedescribed examples and embodiments and are to be included within thescope of the following claims.

What is claimed is:
 1. A composition comprising: a first componentselected from the group consisting of glycosaminoglycans; and a secondcomponent selected from the group consisting of i) polyglycols, ii) longchain hydroxy polyanionic polysaccharides and iii) long chain nitrogencontaining polymers and iii) long chain Nitrogen containing polymers. 2.A composition according to claim 1 wherein the second componentcomprises Sodium Alginate.
 3. A composition according to claim 1 whereinthe second component comprises Alginic Acid.
 4. A composition accordingto claim 1 wherein the second component comprises Propylene GlycolAlginate.
 5. A composition according to claim 1 wherein the secondcomponent comprises Carboxy Methyl cellulose.
 6. A composition accordingto claim 1 wherein the second component comprises Hydroxy Propyl Methylcellulose, Hydroxy Propyl Cellulose, and Methyl Cellulose.
 7. Acomposition according to claim 1 wherein the second component comprisespolylysine, polyhistidine, polyhydroxyl proline, poly ornithine.
 8. Acomposition according to claim 1 wherein the second component comprisespolyethylene glycol.
 9. A composition according to claim 1 wherein thesecond component comprises polylysine.
 10. A composition according toclaim 1 wherein the second component comprises dextran.
 11. Acomposition according to claim 1 wherein the second component compriseshydroxylethyl starch.
 12. A composition according to claim 1 wherein thesecond component comprises chitosan.
 13. A composition according toclaim 1 wherein the second component comprises polyvinyl alcohol.
 14. Acomposition according to claim 1 wherein the second component comprisespolyvinylpyrrolidone.
 15. A composition according to claim 1 wherein thesecond component comprises polyhydroxyprolin.
 16. A compositionaccording to claim 2 wherein the polyglycol has an average molecularweight in the range of about 200 to about 35,000.
 17. A compositionaccording to claim 2 wherein the polyglycol has an average molecularweight in the range of about 6000 to about
 8000. 18. A compositionaccording to claim 2 wherein the polyglycol comprises PEG.
 19. Acomposition according to claim 5 wherein the PEG has an averagemolecular weight in the range of about 200 to about 35,000.
 20. Acomposition according to claim 5 wherein the PEG has an averagemolecular weight in the range of about 6000 to about
 8000. 21. Acomposition according to claim 5 wherein the PEG has an averagemolecular weight of about
 8000. sodium salt of hyaluronic acid.
 22. Amethod for delivering a therapeutic or diagnostic substance to a humanor animal subject, said method comprising the step of combining thetherapeutic or diagnostic substance with a composition that comprises i)a first component selected from the group consisting ofglycosaminoglycans and ii) a second component selected from the groupconsisting of polyglycols and long chain hydroxy polyanionicpolysaccharides.
 23. A method for filling or bulking tissue in a humanor animal subject, said method comprising the step of introducing intothe tissue a filling or bulking amount of a composition that comprisesi) a first component selected from the group consisting ofglycosaminoglycans and ii) a second component selected from the groupconsisting of polyglycols and long chain hydroxy polyanionicpolysaccharides.
 24. A method according to claim 23 wherein thecomposition is injected into the tissue.
 25. A method according to claim24 wherein the composition is injected through a hollow needle, cannulaor other lumen.
 26. A method according to claim 25 wherein thecomposition has a shear thinning grade ratio of 1.50 or greater.
 27. Amethod according to claim 26 wherein the hollow needle, cannula or otherlumen comprises a hypotube or needle of 29 gage or smaller.
 28. A methodaccording to claim 23 wherein the composition is injected as a dermalfiller to fill wrinkles, lines or folds in the skin.
 29. A method formoistening the eye, enhancing corneal wound healing, treating dry eye,treating allergic conjunctivitis or improving the comfort of contactlens wear, said method comprising the step of introducing into the eyean effective amount of a composition that comprises i) a first componentselected from the group consisting of glycosaminoglycans and ii) asecond component selected from the group consisting of polyglycols andlong chain hydroxy polyanionic polysaccharides.